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ana FluorEScEnt tESt ProcEDurE
rEconStItutE BuFFEr (PBS)
1.
Dissolve contents of one buffer pouch in
one liter of deionized or distilled water. The
PBS buffer may be covered and stored at
2-10°C up to four weeks.
DIlutE PatIEnt SamPlES
2.
Screening: Dilute patient samples to 1:40
by adding 0.05 ml (50 µl) serum to 1.95 ml
reconstituted PBS.
Semi-Quantitative Titering: Make serial
dilutions of screening sample(s) (e.g. 1:80, 1:
160, 1:320...1:2560) using PBS.
.
PrEParE SuBStratE SlIDES (20-25 µl/well)
Remove slide(s) from pouch(es) and place
control sera on control wells as follows:
Invert control dropper bottle and squeeze
gently until drop is visible at the tip. Gently
touch the drop to appropriate control well
while avoiding direct contact of dropper
tip with slide surface. Place positive control
on well marked "+", negative control on
well marked "-", and one drop of PBS buffer
on well marked "PBS". Add 1 drop (20-25
µl) patient sample to the numbered wells.
notE: For general screening, the homo-
geneous positive control is recommended.
For semi-quantitative titering, select the
positive control illustrating the most similar
pattern of fluorescence to the screening
sample (e.g. for patient sample yielding a
speckled pattern of fluorescence in screen-
ing, use speckled positive control).
CAUTION: DIRECT CONTACT OF DROPPER
TIP WITH SLIDE SURFACE MAY RESULT IN DAM-
AGE TO THE ANTIGEN SUBSTRATE.
4.
IncuBatE SlIDES (30±5 minutes at room
temperature, i.e. 18-24°C)
Place slide(s) into a moist covered cham-
ber (a petri dish with moistened paper
toweling will be adequate). Incubate, with
lid in place, for 30 minutes (±5 minutes) at
room temperature (18-24°C).
PBS rInSE
5.
Remove slide(s) from incubator tray and
rinse briefly with PBS using a squirt bottle,
Pasteur, or serological pipette. Do not squirt
buffer directly on the wells.
NOTE: To avoid cross contamination on
the slides, direct PBS stream along midline
of slide, tilting first toward the upper row of
wells followed by tilting toward the lower
row of wells.
6.
PBS WaSH (10 minutes)
Wash slide(s) 10 minutes with PBS in a slide
staining dish or Coplin jar. Gentle agita-
tion is recommended at slide immersion,
midpoint, and removal. This wash may be
extended 10-30 minutes with no variability
in final test results. Discard PBS wash solu-
tion after use. For optimal results, change
PBS at midpoint and use a magnetic stirrer.
7.
FluorEScEnt antIBoDY rEagEnt (Cover
the wells with 12-14 drops)
Remove one slide at a time from PBS and
dip 3-5 times in deionized or distilled water.
Tap slide on its side against bibulous paper
or paper toweling to remove excess water.
Immediately return slide to the incubation
chamber and cover the wells completely
using fluorescent antibody reagent; begin
by placing a drop over each well. Repeat
for each slide. Fluorescent antibody re-
agent has been titered to compensate for
residual deionized or distilled water remain-
ing on the slide after rinsing.
notE: It is important that slide wells do not
dry during this procedure or damage to the
substrate may occur.
DO NOT BLOT OR DRY THE SLIDE IN ANY
MANNER OR ALLOW SLIDE TO SIT WITHOUT
FLUORESCENT ANTIBODY REAGENT FOR
LONGER THAN 15 SECONDS.
8.
IncuBatE SlIDES (30±5 minutes at room
temperature, i.e. 18-24°C)
Place lid on incubation chamber and cov-
er with a paper towel to prevent exposure
to light if the chamber is not opaque. Allow
slide(s) to incubate 30 minutes (±5 minutes)
at room temperature (18-24°C).
PBS rInSE
9.
Remove slide(s) from incubator tray and
rinse briefly with PBS. Do not squirt buffer
directly on the wells.
10. PBS WaSH (10 minutes)
Wash slide(s) 10 minutes with PBS in a slide
staining dish or Coplin jar. Gentle agita-
tion is recommended at slide immersion,
midpoint, and removal. This wash may be
extended 10-30 minutes with no variability
in final test results when counterstain is not
used.
Optional Counterstain: (Catalog No. 1105.
Ready-to-use dropper vial containing 5.0 ml
Evans blue solution (0.5% in PBS)).
Add 5-10 drops counterstain per 100 ml
PBS prior to slide immersion. Because the
degree of counterstaining desired may vary
among individuals, counterstaining intensity
may be increased or decreased by simply
adjusting the number of drops added to
the PBS in this wash.
11. mount coVErSlIP
Remove one slide at a time from PBS and
dip 3-5 times in deionized or distilled water.
Tap slide on its side against bibulous paper
or paper toweling to remove excess water.
DO NOT BLOT OR DRY THE SLIDE IN ANY
MANNER OR ALLOW TO SIT WITHOUT COV-
ERSLIP FOR LONGER THAN 15 SECONDS.
Add 4-5 drops of semi-permanent mounting
medium along midline of each slide. Care-
fully place coverslip in position, avoiding air
pockets, by gently lowering coverslip from
one end of the slide to the other.
notE: Excess mounting medium on slide
may result in high background fluores-
cence, due to light scattering, or lack of
clear resolution of cells (blurred image).
Excess mounting medium may be removed
from slide by gently blotting coverslip with
bibulous or lens paper while avoiding any
direct movement of the coverslip.
For Technical Assistance:
1-800-251-5115 (Toll Free) or e-mail:
techservice@immunoconcepts.com
8
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